Rotaviruses (RVs) are ubiquitous, highly infectious segmented dsRNA viruses of importance because they are the most common cause of severe gastroenteritis in children. We will tackle two critical questions in rotavirology regarding how the host innate and adaptive immune systems regulate infection. Our aims are to: 1) Identify the mechanisms by which RV proteins VP3 and NSP1 inhibit innate immune responses. The failure of heterologous (not native to the infected species) RV VP3 and NSP1 proteins to restrict innate responses limits their replication capacity in vitro and vivo. We hypothesize that VP3 mediated degradation of MAVS and NSP1 mediated inhibition of STAT1 are critical determinants of RV host range-restriction (HRR). Preliminary findings reveal that during infection VP3 mediates proteasomal degradation of MAV in vitro and in a HRR manner in vivo. We will dissect the underlying mechanisms of VP3-induced MAVS degradation and its implication for IFN-restricted RV replication in vitro and in vivo. RV protein NSP1 is an archetypal antagonist of IFN induction. RVs inhibit STAT1 activation, a key step in the IFN amplification program, in uninfected cells as well as infected cells in an NSP1-dependent manner. Exciting preliminary data now suggests that STAT1 activation is blocked by NSP1-mediated down-regulation of IFN receptor (IFNR) levels. We will elucidate the mechanisms of RV- mediated inhibition of STAT1 function and IFNR down regulation in infected and bystander cells in vitro and in the context of homologous and heterologous RV infection suckling mice. 2) Identify and characterize targets of heterotypic humoral immunity to RV. The molecular basis for ?heterotypic? immunity to RV in people has remained an enigma for more than forty years. We hypothesize that VP4-cross-reactive antibodies induced after RV infection or monotypic vaccination are the key to developing broad protective immunity and prevention of recurrent severe RV disease. Novel and exciting preliminary data suggests that people can circumvent the substantial serotypic diversity of circulating RVs by generating broadly neutralizing immunoglobulin (Ig) molecules directed at the RV surface protein, VP4. We propose to use a novel strategy to systematically isolate a representative library of monoclonal Igs (mAbs) directed at both RV surface proteins (VP4 and VP7), characterize them at the protein, functional, serotypic, and structural level, and determine their frequency to and specificity for VP4 or VP7 in adults and children after RV infection or vaccination. In addition, we will take advantage of an innovative human intestinal organoid system to test a long standing hypothesis, based on past murine experiments, that ?non-neutralizing? dimeric IgA mAbs directed at the antigenically conserved internal RV VP6 protein can also restrict RV replication in a heterotypic fashion during intestinal epithelial cell (IEC) transcytosis.